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Abstract:

The present invention relates, in general, to attenuated negative-strand
RNA viruses having an impaired ability to antagonize the cellular
interferon (IFN) response, and the use of such attenuated viruses in
vaccine and pharmaceutical formulations. The invention also relates to
the development and use of IFN-deficient systems for selection of such
attenuated viruses.
In particular, the invention relates to attenuated influenza viruses
having modifications to the NS1 gene that diminish or eliminate the
ability of the NS1 gene product to antagonize the cellular IFN response.
The mutant viruses replicate in vivo but demonstrate reduced
pathogenicity, and therefore are well suited for live virus vaccines, and
pharmaceutical formulations.

Claims:

1.-51. (canceled)

52. A genetically engineered attenuated influenza virus, the genome of
which encodes a truncated NS1 protein composed of between 90 and 130
N-terminal amino acid residues of the NS1 protein of the same or a
different influenza virus strain, so that the genetically engineered
influenza virus has an impaired interferon antagonist phenotype.

53. The genetically engineered attenuated influenza virus of claim 52 in
which the genetically engineered attenuated influenza virus genome
encodes a truncated NS1 protein of between 90 and 100 or 100 and 110
N-terminal amino acid residues of the NS1 protein of the same or a
different influenza virus strain.

54. A genetically engineered attenuated influenza virus, the genome of
which encodes a truncated NS1 protein composed of amino acid residues 1
to 130, amino acid residues 1 to 120, amino acid residues 1 to 110, amino
acid residues 1 to 100, amino acid residues 1 to 90, amino acid residues
1 to 89, amino acid residues 1 to 70, or amino acid residues 1 to 60 of
the NS1 protein of the same or a different influenza virus strain, so
that the genetically engineered influenza virus has an impaired
interferon antagonist phenotype.

56. A vaccine formulation comprising a genetically engineered attenuated
chimeric influenza virus, the genome of which encodes a truncated NS1
protein of between 60 and 70, 110 and 120, or 120 and 130 N-terminal
amino acid residues of an NS1 protein of the same or a different
influenza virus strain and which comprises an influenza virus gene
segment encoding a heterologous sequence, wherein the genetically
engineered influenza virus has an impaired phenotype, and physiologically
acceptable excipient.

57. A method for inducing an immune response against an influenza virus,
comprising administering to a subject an effective amount of the vaccine
formulation of claim 56.

58. The vaccine formulation of claim 56, wherein the heterologous
sequence is an antigen of an influenza virus strain variant.

59. The vaccine formulation of claim 56, wherein the influenza virus gene
segment used is the hemagglutinin or neuraminidase gene segment.

60. A method for inducing an immune response against an influenza virus,
comprising administering to a subject an effective amount of a vaccine
formulation, wherein the vaccine formulation comprises the genetically
engineered attenuated influenza virus of claim 52, and a physiologically
acceptable excipient.

61. The method of claim 60, wherein the genetically engineered attenuated
influenza virus genome encodes a truncated NS1 protein of between 90 and
100 or 100 and 110 N-terminal amino acid residues of an NS1 protein of
the same or a different influenza virus strain.

64. A method for inducing an interferon response in a subject having or
at risk for an interferon-sensitive disease comprising administering to a
subject an effective amount of the pharmaceutical composition of claim
63.

65. A recombinant DNA comprising the coding sequence of an influenza
virus NS1 gene, wherein the NS1 gene encodes a truncated NS1 protein
composed of between 90 and 130 N-terminal amino acid residues of the NS1
protein of the same or a different influenza virus strain, so that the
NS1 protein has an impaired interferon antagonist phenotype.

66. The recombinant DNA of claim 65, wherein the NS1 gene encodes a
truncated NS1 protein or between 90 and 100 or 100 and 110 N-terminal
amino acid residues of the NS1 protein of the same or a different
influenza virus strain.

68. A method for generating an attenuated influenza virus comprising: (a)
introducing the recombinant DNA of claim 65 into a cell that provides the
other viral segments and the viral proteins required to produce viral
particles; and (b) culturing said cells, wherein the attenuated influenza
is produced.

69. A method for treating tumors in a subject, comprising administering
to the subject a pharmaceutical formulation, wherein the pharmaceutical
formulation comprises a genetically engineered attenuated influenza
virus, the genome of which encodes a truncated NS1 protein, so that the
genetically engineered influenza virus has an impaired interferon
antagonist phenotype, and physiologically acceptable excipient.

Description:

[0001] This application is a divisional of, and claims benefit of,
copending U.S. application Ser. No. 12/148,798, which is a continuation
of U.S. application Ser. No. 10/713,732, filed Nov. 14, 2003, which
issued as U.S. Pat. No. 7,588,768 on Sep. 15, 2009, which is a
continuation of, and claims benefit of, U.S. application Ser. No.
09/332,288, filed Jun. 11, 1999, which issued as U.S. Pat. No. 6,669,943
on Dec. 30, 2003, which is a continuation-in-part of Application Ser. No.
60/117,683 filed Jan. 29, 1999; Application Ser. No. 60/108,832 filed
Nov. 18, 1998; and Application Ser. No. 60/089,103 filed Jun. 12, 1998,
each of which is incorporated by reference in its entirety herein.

1. INTRODUCTION

[0003] The present invention relates, in general, to attenuated
negative-strand RNA viruses having an impaired ability to antagonize the
cellular interferon (IFN) response, and the use of such attenuated
viruses in vaccine and pharmaceutical formulations. The invention also
relates to the development and use of IFN-deficient systems for the
selection, identification and propagation of such attenuated viruses.

[0004] In a particular embodiment, the invention relates to attenuated
influenza viruses having modifications to the NS1 gene that diminish or
eliminate the ability of the NS1 gene product to antagonize the cellular
IFN response. The mutant viruses replicate in vivo, but demonstrate
reduced pathogenicity, and therefore are well suited for use in live
virus vaccines, and pharmaceutical formulations.

2. BACKGROUND OF THE INVENTION

2.1 The Influenza Virus

[0005] Virus families containing enveloped single-stranded RNA of the
negative-sense genome are classified into groups having non-segmented
genomes (Paramyxoviridae, Rhabdoviridae, Filoviridae and Borna Disease
Virus) or those having segmented genomes (Orthomyxoviridae, Bunyaviridae
and Arenaviridae). The Orthomyxoviridae family, described in detail
below, and used in the examples herein, includes the viruses of
influenza, types A, B and C viruses, as well as Thogoto and Dhori viruses
and infectious salmon anemia virus.

[0006] The influenza virions consist of an internal ribonucleoprotein core
(a helical nucleocapsid) containing the single-stranded RNA genome, and
an outer lipoprotein envelope lined inside by a matrix protein (M1). The
segmented genome of influenza A virus consists of eight molecules (seven
for influenza C) of linear, negative polarity, single-stranded RNAs which
encode ten polypeptides, including: the RNA-dependent RNA polymerase
proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the
nucleocapsid; the matrix membrane proteins (M1, M2); two surface
glycoproteins which project from the lipid containing envelope:
hemagglutinin (HA) and neuraminidase (NA); the nonstructural protein
(NS1) and nuclear export protein (NEP). Transcription and replication of
the genome takes place in the nucleus and assembly occurs via budding on
the plasma membrane. The viruses can reassort genes during mixed
infections.

[0007] Influenza virus adsorbs via HA to sialyloligosaccharides in cell
membrane glycoproteins and glycolipids. Following endocytosis of the
virion, a conformational change in the HA molecule occurs within the
cellular endosome which facilitates membrane fusion, thus triggering
uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is
transcribed. Viral mRNA is transcribed by a unique mechanism in which
viral endonuclease cleaves the capped 5'-terminus from cellular
heterologous mRNAs which then serve as primers for transcription of viral
RNA templates by the viral transcriptase. Transcripts terminate at sites
15 to 22 bases from the ends of their templates, where oligo(U) sequences
act as signals for the addition of poly(A) tracts. Of the eight viral RNA
molecules so produced, six are monocistronic messages that are translated
directly into the proteins representing HA, NA, NP and the viral
polymerase proteins, PB2, PB1 and PA. The other two transcripts undergo
splicing, each yielding two mRNAs which are translated in different
reading frames to produce M1, M2, NS1 and NEP. In other words, the eight
viral RNA segments code for ten proteins: nine structural and one
nonstructural. A summary of the genes of the influenza virus and their
protein products is shown in Table I below.

[0009] Inactivated virus vaccines are prepared by "killing" the viral
pathogen, e.g., by heat or formalin treatment, so that it is not capable
of replication. Inactivated vaccines have limited utility because they do
not provide long lasting immunity and, therefore, afford limited
protection. An alternative approach for producing virus vaccines involves
the use of attenuated live virus vaccines. Attenuated viruses are capable
of replication but are not pathogenic, and, therefore, provide for longer
lasting immunity and afford greater protection. However, the conventional
methods for producing attenuated viruses involve the chance isolation of
host range mutants, many of which are temperature sensitive; e.g., the
virus is passaged through unnatural hosts, and progeny viruses which are
immunogenic, yet not pathogenic, are selected.

[0010] A conventional substrate for isolating and growing influenza
viruses for vaccine purposes are embryonated chicken eggs. Influenza
viruses are typically grown during 2-4 days at 37° C. in 10-11 day
old eggs. Although most of the human primary isolates of influenza A and
B viruses grow better in the amniotic sac of the embryos, after 2 to 3
passages the viruses become adapted to grow in the cells of the allantoic
cavity, which is accessible from the outside of the egg (Murphy, B. R.,
and R. G. Webster, 1996. Orthomyxoviruses p. 1397-1445. In Fields
Virology. Lippincott-Raven P. A.).

[0011] Recombinant DNA technology and genetic engineering techniques, in
theory, would afford a superior approach to producing an attenuated virus
since specific mutations could be deliberately engineered into the viral
genome. However, the genetic alterations required for attenuation of
viruses are not known or predictable. In general, the attempts to use
recombinant DNA technology to engineer viral vaccines have mostly been
directed to the production of subunit vaccines which contain only the
protein subunits of the pathogen involved in the immune response,
expressed in recombinant viral vectors such as vaccinia virus or
baculovirus. More recently, recombinant DNA techniques have been utilized
in an attempt to produce herpes virus deletion mutants or polioviruses
which mimic attenuated viruses found in nature or known host range
mutants. Until 1990, the negative strand RNA viruses were not amenable to
site-specific manipulation at all, and thus could not be genetically
engineered.

[0012] Attenuated live influenza viruses produced thus far might not be
capable of suppressing the interferon response in the host in which they
replicate. Therefore, although these viruses are beneficial because they
are immunogenic and not pathogenic, they are difficult to propagate in
conventional substrates for the purposes of making vaccines. Furthermore,
attenuated viruses may possess virulence characteristics that are so mild
as to not allow the host to mount an immune response sufficient to meet
subsequent challenges.

3. SUMMARY OF THE INVENTION

[0013] The present invention relates to attenuated negative strand RNA
viruses having an impaired ability to antagonize the cellular IFN
response, and the use of such viruses in vaccine and pharmaceutical
formulations. The mutant viruses with an impaired IFN antagonist activity
are attenuated--they are infectious, can replicate in vivo to provide
subclinical levels of infection, and are not pathogenic. Therefore, they
are ideal candidates for live virus vaccines. Moreover, the attenuated
viruses can induce a robust IFN response which has other biological
consequences in vivo, affording protection against subsequent infectious
diseases and/or inducing antitumor responses. Therefore, the attenuated
viruses can be used pharmaceutically, for the prevention or treatment of
other infectious diseases, cancer in high risk individuals, and/or
IFN-treatable diseases.

[0014] The negative strand RNA viruses used in accordance with the
invention include both segmented and non-segmented viruses; preferred
embodiments include but are not limited to influenza virus, respiratory
syncytial virus (RSV), Newcastle disease virus (NDV), vesicular
stomatitis virus (VSV), and parainfluenza virus (PIV). The viruses used
in the invention may be selected from naturally occurring strains,
variants or mutants; mutagenized viruses (e.g., generated by exposure to
mutagens, repeated passages and/or passage in non-permissive hosts);
reassortants (in the case of segmented viral genomes); and/or genetically
engineered viruses (e.g. using the "reverse genetics" techniques) having
the desired phenotype--i.e., an impaired ability to antagonize the
cellular IFN response. The mutant or genetically engineered virus can be
selected based on differential growth in IFN deficient systems versus IFN
competent systems. For example, viruses which grow in an IFN deficient
system, but not in an IFN competent system (or which grow less well in an
IFN competent system) can be selected.

[0015] The attenuated virus so selected can itself be used as the active
ingredient in vaccine or pharmaceutical formulations. Alternatively, the
attenuated virus can be used as the vector or "backbone" of recombinantly
produced vaccines. To this end, the "reverse genetics" technique can be
used to engineer mutations or introduce foreign epitopes into the
attenuated virus, which would serve as the "parental" strain. In this
way, vaccines can be designed for immunization against strain variants,
or in the alternative, against completely different infectious agents or
disease antigens. For example, the attenuated virus can be engineered to
express neutralizing epitopes of other preselected strains.
Alternatively, epitopes of viruses other than negative strand RNA viruses
can be built into the attenuated mutant virus (e.g., gp160, gp120, or
gp41 of HIV). Alternatively, epitopes of non-viral infectious pathogens
(e.g., parasites, bacteria, fungi) can be engineered into the virus. In
yet another alternative, cancer vaccines can be prepared, e.g. by
engineering tumor antigens into the attenuated viral backbone.

[0016] In a particular embodiment involving RNA viruses with segmented
genomes, reassortment techniques can be used to transfer the attenuated
phenotype from a parental segmented RNA virus strain (a natural mutant, a
mutagenized virus, or a genetically engineered virus) to a different
virus strain (a wild-type virus, natural mutant, a mutagenized virus, or
a genetically engineered virus).

[0017] The attenuated viruses, which induce robust IFN responses in hosts,
may also be used in pharmaceutical formulations for the prophylaxis or
treatment of other viral infections, or IFN-treatable diseases, such as
cancer. In this regard, the tropism of the attenuated virus can be
altered to target the virus to a desired target organ, tissue or cells in
vivo or ex vivo. Using this approach, the IFN response can be induced
locally, at the target site, thus avoiding or minimizing the side effects
of systemic IFN treatments. To this end, the attenuated virus can be
engineered to express a ligand specific for a receptor of the target
organ, tissue or cells.

[0018] The invention is based, in part, on the Applicants' discovery that
NS1 of wild type influenza virus functions as an IFN antagonist, in that
NS1 inhibits the IFN mediated response of virus-infected host cells.
Viral mutants deficient for NS1 activity were found to be potent inducers
of the cellular IFN response, and demonstrated an attenuated phenotype in
vivo; i.e. the mutant viruses replicate in vivo, but have reduced
pathogenic effects. While not intending to be bound to any theory or
explanation for how the invention works, the attenuated features of the
viruses of the invention are presumably due to their ability to induce a
robust cellular IFN response, and their impaired ability to antagonize
the host IFN response. However, the beneficial features of the attenuated
viruses of the invention may not be solely attributable to the effects on
the cellular interferon response. Indeed, alterations in other activities
associated with NS1 may contribute to the desired attenuated phenotype.

[0019] The mutant influenza viruses with impaired IFN antagonist activity
were shown to replicate in vivo generating titers that are sufficient to
induce immunological and cytokine responses. For example, vaccination
with attenuated influenza virus reduced viral titer in animals that were
subsequently challenged with wild-type influenza virus. The attenuated
influenza viruses also demonstrated antiviral and antitumor activity.
Pre-infection with attenuated influenza virus inhibited replication of
other strains of wild type influenza virus, and other viruses (such as
Sendai virus) superinfected in embryonated eggs. Inoculation of the
attenuated influenza in animals injected with tumor cells reduced the
number of foci formed. Because influenza virus is known to induce a CTL
(cytotoxic T lymphocyte) response, the attenuated virus is a very
attractive candidate for cancer vaccines.

[0020] Mutations which diminish but do not abolish the IFN antagonist
activity of the virus are preferred for vaccine formulations--such
viruses can be selected for growth in both conventional and
non-conventional substrates, and for intermediate virulence. In
particular, the Applicants have demonstrated that an NS1
C-terminal-truncation mutant replicates to high titers in IFN deficient
substrates, such as 6 and 7-day-old embryonated chicken eggs, as well as
in the allantoic membrane of 10-day-old embryonated chicken eggs, the
conventional substrate for influenza virus that does not permit the
growth of influenza virus mutants in which the entire NS1 gene is deleted
(also referred to herein as "knockout" mutants). However, replication of
the NS1-C terminal truncation mutant is diminished in 12-day-old
embryonated eggs. This approach allows, for the first time, the
generation and identification of live attenuated negative strand RNA
viruses that have altered, but not abolished, IFN antagonist activity,
and that are able to grow in substrates suitable for vaccine preparation.
This approach also allows for the first time, an efficient selection
identification system for influenza or other viruses which contain
mutations that confer altered, but not abolished, interferon antagonist
activity.

[0021] The invention also relates to the use of IFN deficient systems to
propagate the attenuated viruses that cannot be grown in conventional
systems currently used for vaccine production. The term "IFN-deficient
systems" as used herein refers to systems, e.g., cells, cell lines and
animals, such as mice, chickens, turkeys, rabbits, rats, etc., which do
not produce IFN or produce low levels of IFN, do not respond or respond
less efficiently to IFN, and/or are deficient in the activity of
antiviral genes induced by IFN. To this end, Applicants have identified
or designed a number of IFN-deficient systems that can be used, including
but not limited to young embryonated eggs, IFN-deficient cell lines (such
as VERO cells or genetically engineered cell lines such as STAT1
knockouts). Alternatively, embryonated eggs or cell lines can be
pretreated with compounds that inhibit the IFN system (including drugs,
antibodies, antisense, ribozymes, etc.). Yet another embodiment involves
the use of eggs deficient in the IFN system, e.g., eggs produced by STAT1
negative birds, especially fowl, including but not limited to transgenic
chickens, ducks or turkeys.

4. DESCRIPTION OF THE FIGURES

[0022]FIG. 1. DelNS1 virus inhibits wild-type influenza A virus
replication in eggs. Ten-day-old embryonated chicken eggs were inoculated
with the indicated pfu of delNS1 virus. Eight hours later, the eggs were
infected with 103 pfu of WSN virus. After two days of incubation at
37° C., the allantoic fluid was harvested and WSN virus titers
were determined by plaque assay in MDBK cells. Results are the average of
two eggs.

[0023]FIG. 2. Induction of an antiviral response in embryonated eggs by
delNS1 virus. Ten-day-old embryonated chicken eggs were inoculated with
PBS (untreated) or with 2×104 pfu of delNS1 virus (delNS1
treated). Eight hours later, the eggs were now infected with 103 pfu
of influenza A/WSN/33 (H1N1) virus, influenza A/PR8/34 (H+N1) virus,
influenza A/X-31 (H3N2) virus, influenza B/Lee/40 virus, or Sendai virus.
After two days of incubation, the allantoic fluid was harvested and virus
titers were determined by a hemagglutination assay. Results are the
average of two eggs.

[0024] FIG. 3. CV1 cells were transfected with a plasmid expressing IRF-3
fused to the green fluorescent protein (GFP). This allowed determining
the localization of IRF-3 inside the cells by fluorescence microscopy. In
some cases, an NS1 expression plasmid was cotransfected with the IRF-3
expression plasmid at the indicated ratios. 24 hours posttransfection
cells were infected at high moi with PR8(WT) or with delNS1 virus as
indicated. 10 hours postinfection, cells were analyzed in a fluorescence
microscope for IRF-3-GFP localization. The percentage of cells showing
exclusive cytoplasmic localization (CYT) and both cytoplasmic and nuclear
localizations of IRF-3 (Nuc+Cyt) are indicated.

5. DETAILED DESCRIPTION OF THE INVENTION

[0025] The invention relates to the generation, selection and
identification of attenuated negative strand RNA viruses that have an
impaired ability to antagonize the cellular IFN response, and the use of
such viruses in vaccine and pharmaceutical formulations.

[0026] The viruses can have segmented or non-segmented genomes and can be
selected from naturally occurring strains, variants or mutants;
mutagenized viruses (e.g., by exposure to UV irradiation, mutagens,
and/or passaging); reassortants (for viruses with segmented genomes);
and/or genetically engineered viruses. For example, the mutant viruses
can be generated by natural variation, exposure to UV irradiation,
exposure to chemical mutagens, by passaging in non-permissive hosts, by
reassortment (i.e., by coinfection of an attenuated segmented virus with
another strain having the desired antigens), and/or by genetic
engineering (e.g., using "reverse genetics"). The viruses selected for
use in the invention have defective IFN antagonist activity and are
attenuated; i.e., they are infectious and can replicate in vivo, but only
generate low titers resulting in subclinical levels of infection that are
non-pathogenic. Such attenuated viruses are ideal candidates for live
vaccines.

[0027] In a preferred embodiment, the attenuated viruses selected for use
in the invention should be capable of inducing a robust IFN response in
the host--a feature which contributes to the generation of a strong
immune response when used as a vaccine, and which has other biological
consequences that make the viruses useful as pharmaceutical agents for
the prevention and/or treatment of other viral infections, or tumor
formation in high risk individuals, or other diseases which are treated
with IFN.

[0028] The invention is based, in part, on a number of discoveries and
observations made by the Applicants when working with influenza virus
mutants. However, the principles can be analogously applied and
extrapolated to other segmented and non-segmented negative strand RNA
viruses including, but not limited to paramyxoviruses (Sendai virus,
parainfluenza virus, mumps, Newcastle disease virus), morbillivirus
(measles virus, canine distemper virus and rinderpest virus); pneumovirus
(respiratory syncytial virus and bovine respiratory virus); and
rhabdovirus (vesicular stomatitis virus and lyssavirus).

[0029] First, the IFN response is important for containing viral infection
in vivo. The Applicants found that growth of wild-type influenza virus
A/WSN/33 in IFN-deficient mice (STAT1-/- mice) resulted in pan-organ
infection; i.e., viral infection was not confined to the lungs as it is
in wild-type mice which generate an IFN response (Garcia-Sastre, et al.,
1998, J. Virol. 72:8550, which is incorporated by reference herein in its
entirety). Second, the Applicants established that NS1 of influenza virus
functions as an IFN antagonist. The Applicants discovered that an
influenza virus mutant deleted of the entire NS1 gene (i.e., an NS1
"knockout") was not able to grow to high titers in IFN-competent host
cells, and could only be propagated in IFN-deficient hosts. The NS1
knockout virus demonstrated an attenuated phenotype (i.e., it was lethal
in IFN deficient STAT1-/- mice, but not in wild-type mice) and was found
to be a potent inducer of IFN responses in host cells (Garcia-Sastre, et
al., 1998, Virology 252:324-330, which is incorporated by reference
herein in its entirety). Preinfection with the NS1 knockout mutant virus
reduced titers of wild-type influenza and other viruses (e.g., Sendai)
superinfected in embryonated eggs. In another experiment, infection with
the NS1 knockout mutant influenza virus reduced foci formation in animals
inoculated with tumor cells. Thus, the NS1 knockout influenza virus
demonstrated interesting biological properties. However, the NS1 knockout
mutant viruses could not be propagated in conventional systems for
vaccine production. To overcome this problem, the Applicants used and
developed IFN-deficient systems that allow for production of reasonable
yields of attenuated virus.

[0030] In addition, the Applicants designed deletion mutants of NS1, which
do not delete the entire gene. Surprisingly, these NS1 mutants were found
to display an "intermediate" phenotype--the virus can be grown in
conventional hosts used for propagating influenza virus (although growth
is better in the IFN-deficient systems which yield higher titers). Most
importantly, the deletion mutants are attenuated in vivo, and induce a
robust IFN response. Vaccination with the influenza virus NS1 truncated
mutants resulted in low titers of virus in animals subsequently
challenged with wild-type virus, and afforded protection against disease.

[0031] The present invention also relates to the substrates designed for
the isolation, identification and growth of viruses for vaccine purposes.
In particular, interferon-deficient substrates for efficiently growing
influenza virus mutants are described. In accordance with the present
invention, an interferon-deficient substrate is one that is defective in
its ability to produce or respond to interferon.

[0032] The substrate of the present invention may be used for the growth
of any number of viruses which may require interferon-deficient growth
environment. Such viruses may include, but are not limited to
paramyxoviruses (Sendai virus, parainfluenza virus, mumps, Newcastle
disease virus), morbillivirus (measles virus, canine distemper virus and
rinderpest virus); pneumovirus (respiratory syncytial virus and bovine
respiratory virus); rhabdovirus (vesicular stomatitis virus and
lyssavirus).

[0033] The invention also relates to the use of the attenuated virus of
the invention in vaccines and pharmaceutical preparations for humans or
animals. In particular, the attenuated viruses can be used as vaccines
against a broad range of viruses and/or antigens, including but not
limited to antigens of strain variants, different viruses or other
infectious pathogens (e.g., bacteria, parasites, fungi), or tumor
specific antigens. In another embodiment, the attenuated viruses, which
inhibit viral replication and tumor formation, can be used for the
prophylaxis or treatment of infection (viral or nonviral pathogens) or
tumor formation or treatment of diseases for which IFN is of therapeutic
benefit. Many methods may be used to introduce the live attenuated virus
formulations to a human or animal subject to induce an immune or
appropriate cytokine response. These include, but are not limited to,
intranasal, intratrachial, oral, intradermal, intramuscular,
intraperitoneal, intravenous and subcutaneous routes. In a preferred
embodiment, the attenuated viruses of the present invention are
formulated for delivery intranasally.

5.1 Generation of Mutants with Altered IFN Antagonist Activity

[0034] Any mutant virus or strain which has a decreased IFN antagonist
activity can be selected and used in accordance with the invention. In
one embodiment, naturally occurring mutants or variants, or spontaneous
mutants can be selected that have an impaired ability to antagonize the
cellular IFN response. In another embodiment, mutant viruses can be
generated by exposing the virus to mutagens, such as ultraviolet
irradiation or chemical mutagens, or by multiple passages and/or passage
in non-permissive hosts. Screening in a differential growth system can be
used to select for those mutants having impaired IFN antagonist function.
For viruses with segmented genomes, the attenuated phenotype can be
transferred to another strain having a desired antigen by reassortment,
(i.e., by coinfection of the attenuated virus and the desired strain, and
selection for reassortants displaying both phenotypes).

[0035] In another embodiment, mutations can be engineered into a negative
strand RNA virus such as influenza, RSV, NDV, VSV and PIV, using "reverse
genetics" approaches. In this way, natural or other mutations which
confer the attenuated phenotype can be engineered into vaccine strains.
For example, deletions, insertions or substitutions of the coding region
of the gene responsible for IFN antagonist activity (such as the NS1 of
influenza) can be engineered. Deletions, substitutions or insertions in
the non-coding region of the gene responsible for IFN antagonist activity
are also contemplated. To this end, mutations in the signals responsible
for the transcription, replication, polyadenylation and/or packaging of
the gene responsible or the IFN-antagonist activity can be engineered.
For example, in influenza, such modifications can include but are not
limited to: substitution of the non-coding regions of an influenza A
virus gene by the non-coding regions of an influenza B virus gene
(Muster, et al., 1991, Proc. Natl. Acad. Sci. USA, 88:5177), base pairs
exchanges in the non-coding regions of an influenza virus gene (Fodor, et
al., 1998, J. Virol. 72:6283), mutations in the promoter region of an
influenza virus gene (Piccone, et al., 1993, Virus Res. 28:99; Li, et
al., 1992, J. Virol. 66:4331), substitutions and deletions in the stretch
of uridine residues at the 5' end of an influenza virus gene affecting
polyadenylation (Luo, et al., 1991, J. Virol. 65:2861; Li, et al., J.
Virol. 1994, 68(2):1245-9). Such mutations, for example to the promoter,
could down-regulate the expression of the gene responsible for IFN
antagonist activity. Mutations in viral genes which may regulate the
expression of the gene responsible for IFN antagonist activity are also
within the scope of viruses that can be used in accordance with the
invention.

[0036] The present invention also relates to mutations to the NS1 gene
segment that may not result in an altered IFN antagonist activity or an
IFN-inducing phenotype but rather results in altered viral functions and
an attenuated phenotype e.g., altered inhibition of nuclear export of
poly(A)-containing mRNA, altered inhibition of pre-mRNA splicing, altered
inhibition of the activation of PKR by sequestering of dsRNA, altered
effect on translation of viral RNA and altered inhibition of
polyadenylation of host mRNA (e.g., see Krug in Textbook of Influenza,
Nicholson et al. Ed. 1998, 82-92, and references cited therein).

[0037] The reverse genetics technique involves the preparation of
synthetic recombinant viral RNAs that contain the non-coding regions of
the negative strand virus RNA which are essential for the recognition by
viral polymerases and for packaging signals necessary to generate a
mature virion. The recombinant RNAs are synthesized from a recombinant
DNA template and reconstituted in vitro with purified viral polymerase
complex to form recombinant ribonucleoproteins (RNPs) which can be used
to transfect cells. A more efficient transfection is achieved if the
viral polymerase proteins are present during transcription of the
synthetic RNAs either in vitro or in vivo. The synthetic recombinant RNPs
can be rescued into infectious virus particles. The foregoing techniques
are described in U.S. Pat. No. 5,166,057 issued Nov. 24, 1992; in U.S.
Pat. No. 5,854,037 issued Dec. 29, 1998; in European Patent Publication
EP 0702085A1, published Feb. 20, 1996; in U.S. patent application Ser.
No. 09/152,845; in International Patent Publications PCT WO97/12032
published Apr. 3, 1997; WO96/34625 published Nov. 7, 1996; in European
Patent Publication EP-A780475; WO 99/02657 published Jan. 21, 1999; WO
98/53078 published Nov. 26, 1998; WO 98/02530 published Jan. 22, 1998; WO
99/15672 published Apr. 1, 1999; WO 98/13501 published Apr. 2, 1998; WO
97/06270 published Feb. 20, 1997; and EPO 780 47SA1 published Jun. 25,
1997, each of which is incorporated by reference herein in its entirety.

[0038] Attenuated viruses generated by the reverse genetics approach can
be used in the vaccine and pharmaceutical formulations described herein.
Reverse genetics techniques can also be used to engineer additional
mutations to other viral genes important for vaccine production--i.e.,
the epitopes of useful vaccine strain variants can be engineered into the
attenuated virus. Alternatively, completely foreign epitopes, including
antigens derived from other viral or non-viral pathogens can be
engineered into the attenuated strain. For example, antigens of
non-related viruses such as HIV (gp160, gp120, gp41) parasite antigens
(e.g., malaria), bacterial or fungal antigens or tumor antigens can be
engineered into the attenuated strain. Alternatively, epitopes which
alter the tropism of the virus in vivo can be engineered into the
chimeric attenuated viruses of the invention.

[0039] In an alternate embodiment, a combination of reverse genetics
techniques and reassortant techniques can be used to engineer attenuated
viruses having the desired epitopes in segmented RNA viruses. For
example, an attenuated virus (generated by natural selection, mutagenesis
or by reverse genetics techniques) and a strain carrying the desired
vaccine epitope (generated by natural selection, mutagenesis or by
reverse genetics techniques) can be co-infected in hosts that permit
reassortment of the segmented genomes. Reassortants that display both the
attenuated phenotype and the desired epitope can then be selected.

[0040] In another embodiment, the virus to be mutated is a DNA virus
(e.g., vaccinia, adenovirus, baculovirus) or a positive strand RNA virus
(e.g., polio virus). In such cases, recombinant DNA techniques which are
well known in the art may be used (e.g., see U.S. Pat. No. 4,769,330 to
Paoletti, U.S. Pat. No. 4,215,051 to Smith each of which is incorporated
herein by reference in its entirety).

[0041] Any virus may be engineered in accordance with the present
invention, including but not limited to the families set forth in Table 2
below.

[0042] In a preferred embodiment, the present invention relates to
genetically engineered influenza viruses containing deletions and/or
truncations of the NS1 gene product. NS1 mutants of influenza A and B are
particularly preferred. In one approach, portions of the amino terminal
region of the NS1 gene product are retained whereas portions of the
C-terminal region of the NS1 gene product are deleted. Specific desired
mutations can be engineered by way of nucleic acid insertion, deletion,
or mutation at the appropriate codon. In particular, the truncated NS1
proteins have from 1-60 amino acids, 1-70 amino acids, 1-80 amino acids,
1-90 amino acids (the N-terminal amino acid is 1), and preferably 90
amino acids; from 1-100 amino acids, and preferably 99 amino acids; from
1-110 amino acids; from 1-120 amino acids; or from 1-130 amino acids, and
preferably 124 amino acids of the wildtype NS1 gene product.

[0043] The present invention also relates to any genetically engineered
influenza virus in which the NS1 gene product has been modified by
truncation or modification of the NS1 protein that confers upon the
mutant viruses the following phenotypes: the ability of the viruses to
grow to high titers in unconventional substrates, such as 6-7 day old
chicken eggs, or the ability of the viruses to induce a host interferon
response. For influenza A viruses, these include, but are not limited to:
viruses having an NS1 truncations.

[0044] The present invention includes the use of naturally occurring
mutant influenza viruses A or B having the attenuated phenotype, as well
as influenza virus strains engineered to contain such mutations
responsible for the attenuated phenotype. For influenza A viruses, these
include, but are not limited to: viruses having an NS1 of 124 amino acids
(Norton et al., 1987, Virology 156:204-213, which is incorporated by
reference herein in its entirety). For influenza B viruses, these
include, but are not limited to: viruses having an NS1 truncation mutant
comprising 110 amino acids derived from the N-terminus (B/201) (Norton et
al., 1987, Virology 156:204-213, which is incorporated by reference
herein in its entirety), and viruses having an NS1 truncation mutant
comprising 89 amino acids derived from the N-terminus (B/AWBY-234)
(Tobita et al., 1990, Virology 174:314-19, which is incorporated by
reference herein in its entirety). The present invention encompasses the
use of naturally occurring mutants analogous to NS1/38, NS1/80, NS1/124,
(Egorov, et al., 1998, J. Virol. 72(8):6437-41) as well as the naturally
occurring mutants, A/Turkey/ORE/71, B/201 or B/AWBY-234. The present
invention encompasses genetically engineering any influenza A or B virus
such that the genome of the engineered virus comprises a mutation in the
NS1 gene corresponding to the NS1 mutation found in naturally occurring
mutants NS1/80, NS1/124, A/Turkey/ORE/71, B/201 or AWBY-234, with the
proviso that the present invention does not comprise the following
influenza mutants: A/Turkey/Ore/71, B/201 and AWBY-234 as they occur in
nature.

[0045] The attenuated influenza virus may be further engineered to express
antigens of other vaccine strains (e.g., using reverse genetics or
reassortment). Alternatively, the attenuated influenza viruses may be
engineered, using reverse genetics or reassortment with genetically
engineered viruses, to express completely foreign epitopes, e.g.,
antigens of other infectious pathogens, tumor antigens, or targeting
antigens. Since the NS RNA segment is the shortest among the eight viral
RNAs, it is possible that the NS RNA will tolerate longer insertions of
heterologous sequences than other viral genes. Moreover, the NS RNA
segment directs the synthesis of high levels of protein in infected
cells, suggesting that it would be an ideal segment for insertions of
foreign antigens. However, in accordance with the present invention, any
one of the eight segments of influenza viruses may be used for the
insertion of heterologous sequences. For example, where surface antigen
presentation is desired, segments encoding structural proteins, e.g., HA
or NA may be used.

5.2 Host-Restriction Based Selection System

[0046] The invention encompasses methods of selecting viruses which have
the desired phenotype, i.e., viruses which have low or no IFN antagonist
activity, whether obtained from natural variants, spontaneous variants
(i.e., variants which evolve during virus propagation), mutagenized
natural variants, reassortants and/or genetically engineered viruses.
Such viruses can be best screened in differential growth assays that
compare growth in IFN-deficient versus IFN-competent host systems.
Viruses which demonstrate better growth in the IFN-deficient systems
versus IFN competent systems are selected; preferably, viruses which grow
to titers at least one log greater in IFN-deficient systems as compared
to an IFN-competent system are selected.

[0047] Alternatively, the viruses can be screened using IFN assay systems
e.g., transcription based assay systems in which reporter gene expression
is controlled by an IFN-responsive promoter. Reporter gene expression in
infected versus uninfected cells can be measured to identify viruses
which efficiently induce an IFN response, but which are unable to
antagonize the IFN response. In a preferred embodiment, however,
differential growth assays are used to select viruses having the desired
phenotype, since the host system used (IFN-competent versus
IFN-deficient) applies the appropriate selection pressure.

[0048] For example, growth of virus (as measured by titer) can be compared
in a variety of cells, cell lines, or animal model systems that express
IFN and the components of the IFN response, versus cells, cell lines, or
animal model systems deficient for IFN or components of the IFN response.
To this end, growth of virus in cell lines as VERO cells (which are IFN
deficient) versus MDCK cells (which are IFN-competent) can be compared.
Alternatively, IFN-deficient cell lines can be derived and established
from animals bred or genetically engineered to be deficient in the INF
system (e.g., STAT1-/- mutant mice). Growth of virus in such cell lines,
as compared to IFN-competent cells derived, for example, from wild-type
animals (e.g., wild-type mice) can be measured. In yet another
embodiment, cell lines which are IFN-competent and known to support the
growth of wild type virus can be engineered to be IFN-deficient, (e.g.,
by knocking out STAT1, IRF3, PKR, etc.) Techniques which are well known
in the art for the propagation of viruses in cell lines can be used (see,
for example, the working examples infra). Growth of virus in the standard
IFN competent cell line versus the IFN deficient genetically engineered
cell line can be compared.

[0049] Animal systems can also be used. For example, for influenza, growth
in young, IFN-deficient embryonated eggs, e.g., about 6 to about 8 days
old, can be compared to growth in older, IFN-competent eggs, e.g., about
10 to 12 days old. To this end, techniques well known in the art for
infection and propagation in eggs can be used (e.g., see working
examples, infra). Alternatively, growth in IFN-deficient STAT1-/- mice
can be compared to IFN-competent wild type mice. In yet another
alternative, growth in IFN-deficient embryonated eggs produced by, for
example, STAT1-/- transgenic fowl can be compared to growth in
IFN-competent eggs produced by wild-type fowl.

[0050] For purposes of screening, however, transient IFN-deficient systems
can be used in lieu of genetically manipulated systems. For example, the
host system can be treated with compounds that inhibit IFN production
and/or components of the IFN response (e.g., drugs, antibodies against
IFN, antibodies against IFN-receptor, inhibitors of PKR, antisense
molecules and ribozymes, etc.). Growth of virus can be compared in
IFN-competent untreated controls versus IFN-deficient treated systems.
For example, older eggs which are IFN-competent can be pretreated with
such drugs prior to infection with the virus to be screened. Growth is
compared to that achieved in untreated control eggs of the same age.

[0051] The screening methods of the invention provide a simple and easy
screen to identify mutant viruses with abolished IFN antagonist activity
by the inability of the mutant virus to grow in IFN-responsive
environments, as compared to the ability of the mutant virus to grow in
IFN-deficient environments. The screening methods of the invention may
also be used to identify mutant viruses with altered, but not abolished
IFN antagonist activity by measuring the ability of the mutant virus to
grow in both IFN-responsive e.g., 10-day old embryonated eggs or MDCK
cells and IFN-deficient environments e.g., 6-to-7-day old embryonated
eggs or Vero cells. For example, influenza viruses showing at least one
log lower titers in 10-days-old eggs versus 6-7 days old eggs will be
considered impaired in their ability to inhibit the IFN response. In
another example, influenza viruses showing at least one log lower titer
in 12 day old eggs (which mount a high IFN response) versus 10 day old
eggs (which mount a moderate IFN response) are considered partially
impaired in their ability to antagonize the IFN response, and are
considered attractive vaccine candidates.

[0052] The selection methods of the invention also encompass identifying
those mutant viruses which induce IFN responses. In accordance with the
selection methods of the invention, induction of IFN responses may be
measured by assaying levels of IFN expression or expression of target
genes or reporter genes induced by IFN following infection with the
mutant virus or activation of transactivators involved in the IFN
expression and/or the IFN response.

[0053] In yet another embodiment of the selection systems of the
invention, induction of IFN responses may be determined by measuring the
phosphorylated state of components of the IFN pathway following infection
with the test mutant virus, e.g., IRF-3, which is phosphorylated in
response to double-stranded RNA. In response to type I IFN, Jak1 kinase
and TyK2 kinase, subunits of the IFN receptor, STAT1, and STAT2 are
rapidly tyrosine phosphorylated. Thus, in order to determine whether the
mutant virus induces IFN responses, cells, such as 293 cells, are
infected with the test mutant virus and following infection, the cells
are lysed. IFN pathway components, such as Jak1 kinase or TyK2 kinase,
are immunoprecipitated from the infected cell lysates, using specific
polyclonal sera or antibodies, and the tyrosine phosphorylated state of
the kinase is determined by immunoblot assays with an
anti-phosphotyrosine antibody (e.g., see Krishnan et al. 1997, Eur. J.
Biochem. 247: 298-305). An enhanced phosphorylated state of any of the
components of the IFN pathway following infection with the mutant virus
would indicate induction of IFN responses by the mutant virus.

[0054] In yet another embodiment, the selection systems of the invention
encompass measuring the ability to bind specific DNA sequences or the
translocation of transcription factors induced in response to viral
infection, e.g., IRF3, STAT1, STAT2, etc. In particular, STAT1 and STAT2
are phosphorylated and translocated from the cytoplasm to the nucleus in
response to type I IFN. The ability to bind specific DNA sequences or the
translocation of transcription factors can be measured by techniques
known to those of skill in the art, e.g., electromobility gel shift
assays, cell staining, etc.

[0055] In yet another embodiment of the selection systems of the
invention, induction of IFN responses may be determined by measuring
IFN-dependent transcriptional activation following infection with the
test mutant virus. In this embodiment, the expression of genes known to
be induced by IFN, e.g., Mx, PKR, 2-5- oligoadenylatesynthetase, major
histocompatibility complex (MHC) class I, etc., can be analyzed by
techniques known to those of skill in the art (e.g., northern blots,
western blots, PCR, etc.). Alternatively, test cells such as human
embryonic kidney cells or human osteogenic sarcoma cells, are engineered
to transiently or constitutively express reporter genes such as
luciferase reporter gene or chloramphenicol transferase (CAT) reporter
gene under the control of an interferon stimulated response element, such
as the IFN-stimulated promoter of the ISG-54K gene (Bluyssen et al.,
1994, Eur. J. Biochem. 220:395-402). Cells are infected with the test
mutant virus and the level of expression of the reporter gene compared to
that in uninfected cells or cells infected with wild-type virus. An
increase in the level of expression of the reporter gene following
infection with the test virus would indicate that the test mutant virus
is inducing an IFN response.

[0056] In yet another embodiment, the selection systems of the invention
encompass measuring IFN induction by determining whether an extract from
the cell or egg infected with the test mutant virus is capable of
conferring protective activity against viral infection. More
specifically, groups of 10-day old embryonated chicken eggs are infected
with the test mutant virus or the wild-type virus. Approximately 15 to 20
hours post infection, the allantoic fluid is harvested and tested for IFN
activity by determining the highest dilution with protective activity
against VSV infection in tissue culture cells, such as CEF cells.

5.3 Propagation of Virus in Interferon Deficient Growth Substrates

[0057] The invention also encompasses methods and IFN-deficient substrates
for the growth and isolation of naturally occurring or engineered mutant
viruses having altered IFN antagonist activity. IFN-deficient substrates
which can be used to support the growth of the attenuated mutant viruses
include, but are not limited to naturally occurring cells, cell lines,
animals or embryonated eggs that are IFN deficient, e.g., Vero cells,
young embryonated eggs; recombinant cells or cell lines that are
engineered to be IFN deficient, e.g., IFN-deficient cell lines derived
from STAT1 knockout mice or other similarly engineered transgenic
animals; embryonated eggs obtained from IFN deficient birds, especially
fowl (e.g., chickens, ducks, turkeys) including flock that are bred to be
IFN-deficient or transgenic birds (e.g., STAT1 knockouts). Alternatively,
the host system, cells, cell lines, eggs or animals can be genetically
engineered to express transgenes encoding inhibitors of the IFN system,
e.g., dominant-negative mutants, such as STAT1 lacking the DNA binding
domain, antisense RNA, ribozymes, inhibitors of IFN production,
inhibitors of IFN signaling, and/or inhibitors of antiviral genes induced
by IFN. It should be recognized that animals that are bred or genetically
engineered to be IFN deficient will be somewhat immunocompromised, and
should be maintained in a controlled, disease free environment. Thus,
appropriate measures (including the use of dietary antibiotics) should be
taken to limit the risk of exposure to infectious agents of transgenic
IFN deficient animals, such as flocks of breeding hens, ducks, turkeys,
etc. Alternatively, the host system, e.g., cells, cell lines, eggs or
animals can be treated with a compound which inhibits IFN production
and/or the IFN pathway e.g., drugs, antibodies, antisense molecules,
ribozyme molecules targeting the STAT1 gene, and/or antiviral genes
induced by IFN.

[0058] In accordance with the present invention, immature embryonated
chicken eggs encompass eggs which as a course of nature are up to, but
not yet ten-day-old eggs, preferably six-to nine-day-old eggs; and eggs
which artificially mimic immature eggs up to, but not yet ten-day-old, as
a result of alterations to the growth conditions, e.g., changes in
incubation temperatures; treating with drugs; or any other alteration
which results in an egg with a retarded development, such that the IFN
system of the egg is not fully developed as compared to 10- to 12-day-old
eggs.

5.3.1 Natural IFN Deficient Substrates

[0059] In one embodiment, the present invention relates to growing
naturally occurring and engineered mutant viruses in unconventional
substrates, such as immature embryonated eggs which have not yet
developed an IFN system. Immature embryonated eggs are normally not used
to grow virus due to their fragile condition and smaller allantoic
volume. The present invention encompasses growing mutant viruses in
embryonated eggs less than 10 days old; preferably growing mutated virus
in 8-day old embryonated eggs and most preferably, in 6 to 8-day old
eggs.

[0060] The present invention also encompasses methods of growing and
isolating mutated viruses having altered IFN antagonist activity in cells
and cell lines which naturally do not have an IFN pathway or have a
deficient IFN pathway or have a deficiency in the IFN system e.g., low
levels of IFN expression as compared to wild-type cells. In a particular
preferred embodiment, the present invention relates to methods of growing
mutated viruses having an altered IFN antagonist activity in Vero cells.

5.3.2 Genetically Engineered IFN Deficient Substrates

[0061] The present invention relates to methods of growing and isolating
mutated viruses having altered IFN antagonist activity in a genetically
engineered IFN deficient substrate. The present invention encompasses
transgenic avians in which a gene essential to the IFN system is mutated,
e.g., STAT1, which would lay eggs that are IFN deficient. The present
invention further encompasses avian transgenics which express
dominant-negative transcription factors, e.g., STAT1 lacking the DNA
binding domain, ribozymes, antisense RNA, inhibitors of IFN production,
inhibitors of IFN signaling, and inhibitors of antiviral genes induced in
response to IFN. The benefit of using eggs from an IFN-deficient
transgenic avian is that the conventional 10 day age eggs may be used to
grow the virus which are more stable and have a larger volume due to
their larger size. In yet another embodiment, cell lines may be
genetically engineered to be IFN deficient. The present invention
encompasses cell lines in which a gene essential to the IFN synthesis,
IFN pathway, and/or an antiviral gene(s) induced by IFN are mutated,
e.g., STAT1.

[0062] The invention provides recombinant cell lines or animals, in
particular avians, in which one or more genes essential for the IFN
pathway, e.g. interferon receptor, STAT1 etc. has been disrupted, i.e.,
is a "knockout"; the recombinant animal can be any animal but in a
preferred embodiment is an avian, e.g. chicken, turkey, hen, duck, etc.
(see, e.g., Sang, 1994, Trends Biotechnol. 12:415; Perry, et al., 1993,
Transgenic Res. 2:125; Stern, C. D., 1996, Curr Top Microbiol Immunol
212:195-206; and Shuman, 1991, Experientia 47:897 for reviews regarding
the production of avian transgenics each of which is incorporated by
reference herein in its entirety). Such a cell line or animal can be
generated by any method known in the art for disrupting a gene on the
chromosome of the cell or animal. Such techniques include, but are not
limited to pronuclear microinjection (Hoppe & Wagner, 1989, U.S. Pat. No.
4,873,191); retrovirus mediated gene transfer into germ lines (Van der
Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene
targeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313);
electroporation of embryos (Lo, 1983, Mol. Cell. Biol. 3:1803); and
sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717); etc.
For a review of such techniques, see Gordon, 1989, Transgenic Animals,
Intl. Rev. Cytol. 115:171, which is incorporated by reference herein in
its entirety.

[0063] In particular, a STAT1 knockout animal can be produced by promoting
homologous recombination between a STAT1 gene in its chromosome and an
exogenous STAT1 gene that has been rendered biologically inactive
(preferably by insertion of a heterologous sequence, e.g., an antibiotic
resistance gene). Homologous recombination methods for disrupting genes
in the mouse genome are described, for example, in Capecchi (1989,
Science 244:1288) and Mansour et al. (1988, Nature 336:348).

[0064] Briefly, all or a portion of a STAT1 genomic clone is isolated from
genomic DNA from the same species as the knock-out cell or animal. The
STAT1 genomic clone can be isolated by any method known in the art for
isolation of genomic clones (e.g. by probing a genomic library with a
probe derived from a STAT1 sequence such as those sequences provided in
see Meraz et al., 1996, Cell 84:431; Durbin et al. 1996, Cell 84:443, and
references cited therein). Once the genomic clone is isolated, all or a
portion of the clone is introduced into a recombinant vector. Preferably,
the portion of the clone introduced into the vector contains at least a
portion of an exon of the STAT1 gene, i.e., contains a STAT1 protein
coding sequence. A sequence not homologous to the STAT1 sequence,
preferably a positive selectable marker, such as a gene encoding an
antibiotic resistance gene, is then introduced into the STAT1 gene exon.
The selectable marker is preferably operably linked to a promoter, more
preferably a constitutive promoter. The non-homologous sequence is
introduced anywhere in the STAT1 coding sequence that will disrupt STAT1
activity, e.g., at a position where point mutations or other mutations
have been demonstrated to inactivate STAT1 protein function. For example,
but not by way of limitation, the non-homologous sequence can be inserted
into the coding sequence for the portion of the STAT1 protein containing
all or a portion of the kinase domain (e.g., the nucleotide sequence
coding for at least 50, 100, 150, 200 or 250 amino acids of the kinase
domain).

[0065] The positive selectable marker is preferably a neomycin resistance
gene (neo gene) or a hygromycin resistance gene (hygro gene). The
promoter may be any promoter known in the art; by way of example the
promoter may be the phosphoglycerate kinase (PGK) promoter (Adra et al.,
1987, Gene 60:65-74), the PolII promoter (Soriano et al., 1991. Cell
64:693-701), or the MC1 promoter, which is a synthetic promoter designed
for expression in embryo-derived stem cells (Thomas & Capecchi, 1987,
Cell 51:503-512). Use of a selectable marker, such as an antibiotic
resistance gene, allows for the selection of cells that have incorporated
the targeting vector (for example, the expression of the neo gene product
confers resistance to G418, and expression of the hygro gene product
confers resistance to hygromycin).

[0066] In a preferred embodiment, a negative selectable marker for a
counterselection step for homologous, as opposed to non-homologous,
recombination of the vector is inserted outside of the STAT1 genomic
clone insert. For example, such a negative selectable marker is the HSV
thymidine kinase gene (HSV-tk), the expression of which makes cells
sensitive to ganciclovir. The negative selectable marker is preferably
under the control of a promoter such as, but not limited to the PGK
promoter, the PolII promoter or the MC1 promoter.

[0067] When homologous recombination occurs, the portions of the vector
that are homologous to the STAT1 gene, as well as the non-homologous
insert within the STAT1 gene sequences, are incorporated into the STAT1
gene in the chromosome, and the remainder of the vector is lost. Thus,
since the negative selectable marker is outside the region of homology
with the STAT1 gene, cells in which homologous recombination has occurred
(or their progeny), will not contain the negative selectable marker. For
example, if the negative selectable marker is the HSV-tk gene, the cells
in which homologous recombination has occurred will not express thymidine
kinase and will survive exposure to ganciclovir. This procedure permits
the selection of cells in which homologous recombination has occurred, as
compared to non-homologous recombination in which it is likely that the
negative selectable marker is also incorporated into the genome along
with the STAT1 sequences and the positive selectable marker. Thus, cells
in which non-homologous recombination has occurred would most likely
express thymidine kinase and be sensitive to ganciclovir.

[0068] Once the targeting vector is prepared, it is linearized with a
restriction enzyme for which there is a unique site in the targeting
vector, and the linearized vector is introduced into embryo-derived stem
(ES) cells (Gossler et al., 1986, Proc. Natl. Acad. Sci. USA
83:9065-9069) by any method known in the art, for example by
electroporation. If the targeting vector includes a positive selectable
marker and a negative, counterselectable marker, the ES cells in which
homologous recombination has occurred can be selected by incubation in
selective media. For example, if the selectable markers are the neo
resistance gene and the HSV-tk gene, the cells are exposed to G418 (e.g.,
approximately 300 μg/ml) and ganciclovir (e.g., approximately 2
μM).

[0069] Any technique known in the art for genotyping, for example but not
limited to Southern blot analysis or the polymerase chain reaction, can
be used to confirm that the disrupted STAT1 sequences have homologously
recombined into the STAT1 gene in the genome of the ES cells. Because the
restriction map of the STAT1 genomic clone is known and the sequence of
the STAT1 coding sequence is known (see Meraz et al., 1996, Cell 84:431;
Durbin et al., 1996, Cell 84: 443-450, all references cited therein), the
size of a particular restriction fragment or a PCR amplification product
generated from DNA from both the disrupted and non-disrupted alleles can
be determined. Thus, by assaying for a restriction fragment or PCR
product, the size of which differs between the disrupted and
non-disrupted STAT1 gene, one can determine whether homologous
recombination has occurred to disrupt the STAT1 gene.

[0070] The ES cells with the disrupted STAT1 locus can then be introduced
into blastocysts by microinjection and then the blastocysts can be
implanted into the uteri of pseudopregnant mice using routine techniques.
The animal that develop from the implanted blastocysts are chimeric for
the disrupted allele. The chimeric males can be crossed to females, and
this cross can be designed such that germline transmission of the allele
is linked to transmission of a certain coat color. The germline
transmission of the allele can be confirmed by Southern blotting or PCR
analysis, as described above, of genomic DNA isolated from tissue
samples.

5.3.3 Transient IFN Deficient Substrates

[0071] The cells, cell lines, animals or eggs can be pre-treated with
compounds that inhibit the IFN system. In accordance with the present
invention, compounds which inhibit synthesis of IFN, or the activity or
the expression of the components of the IFN system can be used to
pretreat hosts, e.g., compounds that inhibit the synthesis of IFN, the
activity of IFN, the IFN receptor, other targets in the IFN signal
transduction pathway, or that inhibit the activity of antiviral genes
induced by IFN. Examples of compounds which may be used in accordance
with the present invention, include, but are not limited to, nucleic acid
molecules, antibodies, peptides, antagonists of the IFN receptor,
inhibitors of the STAT1 pathway, inhibitors of PKR, etc. In accordance
with the present invention, nucleic acid molecules include antisense
molecules, ribozymes and triple helix molecules that target genes
encoding essential components of the IFN system, e.g., STAT1. Nucleic
acid molecules also encompass nucleotides encoding dominant negative
mutants of components of the IFN system; e.g. prior to infection with the
viral mutant, the cells can be transfected with a DNA encoding a
truncated, signalling incompetent mutant of the IFN receptor.

[0073] The invention encompasses vaccine formulations comprising the
attenuated negative strand RNA viruses having an impaired ability to
antagonize the cellular IFN response, and a suitable excipient. The virus
used in the vaccine formulation may be selected from naturally occurring
mutants or variants, mutagenized viruses or genetically engineered
viruses. Attenuated strains of segmented RNA viruses can also be
generated via reassortment techniques, or by using a combination of the
reverse genetics approach and reassortment techniques. Naturally
occurring variants include viruses isolated from nature as well as
spontaneous occurring variants generated during virus propagation, having
an impaired ability to antagonize the cellular IFN response. The
attenuated virus can itself be used as the active ingredient in the
vaccine formulation. Alternatively, the attenuated virus can be used as
the vector or "backbone" of recombinantly produced vaccines. To this end,
recombinant techniques such as reverse genetics (or, for segmented
viruses, combinations of the reverse genetics and reassortment
techniques) may be used to engineer mutations or introduce foreign
antigens into the attenuated virus used in the vaccine formulation. In
this way, vaccines can be designed for immunization against strain
variants, or in the alternative, against completely different infectious
agents or disease antigens.

[0074] Virtually any heterologous gene sequence may be constructed into
the viruses of the invention for use in vaccines. Preferably, epitopes
that induce a protective immune response to any of a variety of
pathogens, or antigens that bind neutralizing antibodies may be expressed
by or as part of the viruses. For example, heterologous gene sequences
that can be constructed into the viruses of the invention for use in
vaccines include but are not limited to epitopes of human
immunodeficiency virus (HIV) such as gp120; hepatitis B virus surface
antigen (HBsAg); the glycoproteins of herpes virus (e.g. gD, gE); VP1 of
poliovirus; antigenic determinants of non-viral pathogens such as
bacteria and parasites, to name but a few. In another embodiment, all or
portions of immunoglobulin genes may be expressed. For example, variable
regions of anti-idiotypic immunoglobulins that mimic such epitopes may be
constructed into the viruses of the invention. In yet another embodiment,
tumor associated antigens may be expressed.

[0075] Either a live recombinant viral vaccine or an inactivated
recombinant viral vaccine can be formulated. A live vaccine may be
preferred because multiplication in the host leads to a prolonged
stimulus of similar kind and magnitude to that occurring in natural
infections, and therefore, confers substantial, long-lasting immunity.
Production of such live recombinant virus vaccine formulations may be
accomplished using conventional methods involving propagation of the
virus in cell culture or in the allantois of the chick embryo followed by
purification.

[0076] Vaccine formulations may include genetically engineered negative
strand RNA viruses that have mutations in the NS1 or analogous gene
including but not limited to the truncated NS1 influenza mutants
described in the working examples, infra. They may also be formulated
using natural variants, such as the A/turkey/Ore/71 natural variant of
influenza A, or B/201, and B/AWBY-234, which are natural variants of
influenza B. When formulated as a live virus vaccine, a range of about
104 pfu to about 5×106 pfu per dose should be used.

[0077] Many methods may be used to introduce the vaccine formulations
described above, these include but are not limited to intranasal,
intratracheal, oral, intradermal, intramuscular, intraperitoneal,
intravenous, and subcutaneous routes. It may be preferable to introduce
the virus vaccine formulation via the natural route of infection of the
pathogen for which the vaccine is designed, or via the natural route of
infection of the parental attenuated virus. Where a live influenza virus
vaccine preparation is used, it may be preferable to introduce the
formulation via the natural route of infection for influenza virus. The
ability of influenza virus to induce a vigorous secretory and cellular
immune response can be used advantageously. For example, infection of the
respiratory tract by influenza viruses may induce a strong secretory
immune response, for example in the urogenital system, with concomitant
protection against a particular disease causing agent.

[0078] A vaccine of the present invention, comprising
104-5×106 pfu of mutant viruses with altered IFN
antagonist activity, could be administered once. Alternatively, a vaccine
of the present invention, comprising 104-5×106 pfu of
mutant viruses with altered IFN antagonist activity, could be
administered twice or three times with an interval of 2 to 6 months
between doses. Alternatively, a vaccine of the present invention,
comprising 104-5×106 pfu of mutant viruses with altered
IFN antagonist activity, could be administered as often as needed to an
animal, preferably a mammal, and more preferably a human being.

5.5 Pharmaceutical Compositions

[0079] The present invention encompasses pharmaceutical compositions
comprising mutant viruses with altered IFN antagonist activity to be used
as anti-viral agents or anti-tumor agents or as agents against
IFN-sensitive diseases. The pharmaceutical compositions have utility as
an anti-viral prophylactic and may be administered to an individual at
risk of getting infected or is expected to be exposed to a virus. For
example, in the event that a child comes home from school where he is
exposed to several classmates with the flu, a parent would administer the
anti-viral pharmaceutical composition of the invention to herself, the
child and other family members to prevent viral infection and subsequent
illness. People traveling to parts of the world where a certain
infectious disease is prevalent (e.g. hepatitis A virus, malaria, etc.)
can also be treated.

[0080] Alternatively, the pharmaceutical compositions may be used to treat
tumors or prevent tumor formation, e.g., in patients who have cancer or
in those who are at high risk for developing neoplasms or cancer. For
example, patients with cancer can be treated to prevent further
tumorigenesis. Alternatively, subjects who are or are expected to be
exposed to carcinogens can be treated; individuals involved in
environmental cleanups who may be exposed to pollutants (e.g. asbestos)
may be treated. Alternatively, individuals who are to be exposed to
radiation can be treated prior to exposure and thereafter (e.g. patients
exposed to high dose radiation or who must take carcinogenic drugs).

[0081] The use of the attenuated viruses of the invention as antitumor
agents is based on the Applicants' discovery that an attenuated influenza
virus mutant containing a deletion in its IFN-antagonist gene is able to
reduce tumor formation in mice. The antitumor properties of the invention
can be at least partially related to their ability to induce IFN and IFN
responses. Alternatively, the antitumor properties of the attenuated
viruses of the invention can be related to their ability to specifically
grow in and kill tumor cells, many of which are known to have
deficiencies in the IFN system. Regardless of the molecular mechanism(s)
responsible for the antitumor properties, the attenuated viruses of the
invention might be used to treat tumors or to prevent tumor formation.

[0082] The present invention further encompasses the mutant viruses with
an altered IFN-antagonist phenotype which are targeted to specific
organs, tissues and/or cells in the body in order to induce therapeutic
or prophylactic effects locally. One advantage of such an approach is
that the IFN-inducing viruses of the invention are targeted to specific
sites, e.g. the location of a tumor, to induce IFN in a site specific
manner for a therapeutic effect rather than inducing IFN systemically
which may have toxic effects.

[0083] The mutant IFN-inducing viruses of the invention may be engineered
using the methods described herein to express proteins or peptides which
would target the viruses to a particular site. In a preferred embodiment,
the IFN-inducing viruses would be targeted to sites of tumors. In such an
embodiment, the mutant viruses can be engineered to express the antigen
combining site of an antibody which recognized the tumor specific
antigen, thus targeting the IFN-inducing virus to the tumor. In yet
another embodiment, where the tumor to be targeted expresses a hormone
receptor, such as breast or ovarian tumors which express estrogen
receptors, the IFN-inducing virus may be engineered to express the
appropriate hormone. In yet another embodiment, where the tumor to be
targeted expresses a receptor to a growth factor, e.g., VEGF, EGF, or
PDGF, the IFN-inducing virus may be engineered to express the appropriate
growth factor or portion(s) thereof. Thus, in accordance with the
invention, the IFN-inducing viruses may be engineered to express any
target gene product, including peptides, proteins, such as enzymes,
hormones, growth factors, antigens or antibodies, which will function to
target the virus to a site in need of anti-viral, antibacterial,
anti-microbial or anti-cancer activity.

[0084] Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,
epidural, and oral routes. The pharmaceutical compositions of the present
invention may be administered by any convenient route, for example by
infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,
etc.) and may be administered together with other biologically active
agents. Administration can be systemic or local. In addition, in a
preferred embodiment it may be desirable to introduce the pharmaceutical
compositions of the invention into the lungs by any suitable route.
Pulmonary administration can also be employed, e.g., by use of an inhaler
or nebulizer, and formulation with an aerosolizing agent.

[0085] In a specific embodiment, it may be desirable to administer the
pharmaceutical compositions of the invention locally to the area in need
of treatment; this may be achieved by, for example, and not by way of
limitation, local infusion during surgery, topical application, e.g., in
conjunction with a wound dressing after surgery, by injection, by means
of a catheter, by means of a suppository, or by means of an implant, said
implant being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or former site) of
a malignant tumor or neoplastic or pre-neoplastic tissue.

[0087] The pharmaceutical compositions of the present invention comprise a
therapeutically effective amount of the attenuated virus, and a
pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of
the Federal or a state government or listed in the U.S. Pharmacopeia or
other generally recognized pharmacopeiae for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the pharmaceutical composition is
administered. Saline solutions and aqueous dextrose and glycerol
solutions can also be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. These
compositions can take the form of solutions, suspensions, emulsion,
tablets, pills, capsules, powders, sustained-release formulations and the
like. These compositions can be formulated as a suppository. Oral
formulation can include standard carriers such as pharmaceutical grades
of mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by E. W.
Martin. Such compositions will contain a therapeutically effective amount
of the Therapeutic, preferably in purified form, together with a suitable
amount of carrier so as to provide the form for proper administration to
the patient. The formulation should suit the mode of administration.

[0088] The amount of the pharmaceutical composition of the invention which
will be effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques. In addition, in vitro assays
may optionally be employed to help identify optimal dosage ranges. The
precise dose to be employed in the formulation will also depend on the
route of administration, and the seriousness of the disease or disorder,
and should be decided according to the judgment of the practitioner and
each patient's circumstances. However, suitable dosage ranges for
administration are generally about 104-5×106 pfu and can
be administered once, or multiple times with intervals as often as
needed. Pharmaceutical compositions of the present invention comprising
104-5×106 pfu of mutant viruses with altered IFN
antagonist activity, can be administered intranasally, intratracheally,
intramuscularly or subcutaneously. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model test
systems.

6. EXAMPLE

Generation and Characterization of NS1 Truncation Mutants of Influenza A
Virus

[0090] The plasmid pT3NS1/99, which contains a 99 amino acid C-terminal
truncated form of NS1 was made as follows. First, pPUC19-T3/NS PR8,
containing the complete NS gene of PR8 virus flanked by the T3 RNA
polymerase promoter and BpuAI restriction site was amplified by reverse
PCR (Ochman et al., 1988, Genetics 120:621-623) using the appropriate
primers. The obtained cDNA thus containing the truncated NS1 gene was
phosphorylated, Klenow treated, self-ligated and propagated in E. coli
strain TG1. The construct obtained after purification was named pT3NS1/99
and verified by sequencing. Plasmids for expression of NP, PB1, PB2, and
PA proteins of PR8 virus (pHMG-NP, pHMG-PB1, pHMG-PB2, and pHMG-PA) were
previously described (Pleschka et al., 1996, J. Virol. 70:4188-4192).
pPOLI-NS-RB was made by substituting the CAT open reading frame of
pPOLI-CAT-RT (Pleschka et al., 1996, J. Virol. 70:4188-4192) within
RT-PCR product derived from the coding region of the NS gene of influenza
A/WSN/33 (WSN) virus. This plasmid expresses the NS-specific viral RNA
segment of WSN virus under the control of a truncated human polymerase I
promoter.

[0091] Generation of NS1/99 virus was performed by ribonucleoprotein (RNP)
transfection (Luytjes et al., 1989, Cell 59:1107-1113). The RNPs were
formed by T3 RNA polymerase transcription from pT3NS1/99 linearized with
BpuAI in the presence of purified nucleoprotein and polymerase of
influenza 25A-1 virus (Enami, et al., 1991, J. Virol. 65:2711-2713). RNP
complexes were transfected into Vero

[0092] cells which were previously infected with 25A-1 virus. Transfected
cells were incubated for 18 hours at 37° C., and the supernatant
was passaged twice in Vero cells at 40° C. and plaque purified
three times in Vero cells covered with agar overlay media at 37°
C. The isolated NS1/99 virus was analyzed by RT-PCR using specific
primers. The wild-type transfectant virus was generated as follows: Vero
cells in 35-mm dishes were transfected with plasmids pHMG-NP, pHMG-PB1,
pHMG-PB2, pHMG-PA and pPOLI-NS-RB, as previously described (Pleschka et
al., 1996, J. Virol. 70:4188-4192). Two days post-transfection, cells
were infected with 5×104 pfu of delNSl virus and incubated two
more days at 37° C. Cell supernatant was passaged once in MDCK
cells and twice in chicken embryonated eggs. Transfectant viruses were
cloned by limiting dilution in eggs. Genomic RNA from purified NS1/99
transfectant virus was analyzed by polyacrylamide gel electrophoresis, as
previously described (Zheng et al., 1996, Virology 217:242-251).
Expression of a truncated NS1 protein by NS1/99 virus was verified by
immunoprecipitating labeled infected cell extracts using a rabbit
polyclonal antisera against NS1.

[0093] The allantoic cavity of embryonated chicken eggs, aged 6, 10, and
14 days were inoculated with approximate 103 pfu of PR8, NS1/99, or
delNS1 (in which the entire NS1 gene is deleted) viruses, incubated at
37° C. for two days, and the viruses present in the allantoic
fluid were titrated by hemagglugination (HA) assay.

[0094] Groups of 5 BALB/c mice (Taconic Farms) were inoculated
intranasally with 5×106 pfu, 1.5×105 pfu, or
5×103 pfu of wild-type A/PR/8/34 (PR8) or NS1/99 virus.
Inoculations were performed under anesthesia using 50 μl of MEM
containing the appropriate number of plaque forming units of the
appropriate virus. Animals were monitored daily, and sacrificed when
observed in extremis. In a subsequent experiment, all surviving mice were
challenged four weeks later with a dose of 100LD50 of wild-type PR8
virus. All procedures were in accord with NIH guidelines on care and use
of laboratory animals.

6.2 Results

Attenuation of Influenza A Viruses by NS1 Deletions

[0095] Applicants have previously shown that an influenza A virus in which
the NS1 gene was deleted (delNSl virus) is able to grow to titers of
approximately 107 pfu/ml in cells deficient in type I Interferon
(IFN) production, such as Vero cells. However, this virus was impaired in
its ability to replicate and cause disease in mice (Garcia-Sastre et al.,
1998, Virology 252:324). By contrast, delNSl virus was able to grow in
and kill STAT1-/- mice. These results demonstrated that the NS1 protein
of influenza A virus is a virulence factor involved in the inhibition of
the host antiviral responses mediated by type I IFN. The following
experiments were conducted to determine whether one could generate
influenza viruses with virulence characteristics intermediate between
wild-type and delNSl viruses by deleting portions of the NS1 gene and
whether some of these viruses might have optimal characteristics for
being used as live attenuated vaccines against influenza viruses, i.e.,
stability and an appropriate balance between attenuation, immunogenicity
and growth in substrates suitable for vaccine preparation, such as
embryonated chicken eggs.

[0096] In order to test this hypothesis, an influenza A/PR/8/34 (PR8)
virus was generated in which the NS1 gene has been modified in order to
direct the expression of a truncated NS1 protein containing only 99 amino
acids at the amino terminal in common with the 230 amino acids of the
wild-type NS1 protein. This virus (NS1-99) was obtained by RNP
transfection of an artificially engineered NS gene using 25A-1 helper
virus, as previously described (Garcia-Sastre et al., 1998, Virology
252:324). Analysis of NS1 expression in virus infected cells revealed the
truncated nature of the NS1 protein of the NS1-99 virus.

[0097] The ability of delNS1, NS1-99 and wild-type PR8 viruses to grow in
embryonated chicken eggs of different ages was analyzed. The rationale
for this experiment comes from the fact that the ability of embryonated
eggs to synthesize and to respond to type I IFN under an appropriate
stimulus is age dependent. In fact, both IFN inducibility and
responsiveness start at an age of approximately 10 days, and then
exponentially increase with the age (Sekellick et al. 1990, In Vitro
Cell. Dev. Biol. 26:997; Sekellick & Marcus, 1985 J. Interferon Res.
5:657). Thus, the use of eggs of different ages represents a unique
system to test the ability of different viruses to inhibit IFN responses.
Eggs of 6, 10, and 14 days of age were inoculated with approximately
103 pfu of PR8, NS1-99 or delNS1 viruses, incubated at 37° C.
for 2 days, and the viruses present in the allantoic fluid were titrated
by hemagglutination (HA) assay. As shown in Table 3, whereas wild-type
virus grew to similar HA titers in embryonated eggs of 6, 10 and 14 days
of age, delNS1 only replicated to a detectable HA titer in 6-day-old
eggs. By contrast, NS1-99 virus showed an intermediate behavior between
delNS1 and wild-type viruses, and was able to grow to HA titers similar
to those of wild-type virus in 10-day-old eggs, but not in 14-day-old
eggs.

[0098] The attenuation characteristics of NS1-99 virus were next
determined in mice. For this purpose, groups of 5 BALB/c mice were
intranasally infected with 5×106 pfu, 1.5×105 or
1.5×103 pfu of wild-type PR8 or NS1-99 virus. Mice were then
monitored during 3 weeks for survival. The results are given in Table 4.
NS1-99 virus had an LD50 at least three logs higher than that of
wild-type virus.

[0099] Experimental details are similar to those in Section 6.1. Two
mutant influenza B viruses, B/610B5B/201 (B/201) and B/AWBY-234,
127-amino-acids and 90 amino acids in length (C-terminal truncated NS1
proteins), respectively (Norton et al., 1987 Virology 156:204; Tobita et
al., 1990 Virology 174:314) were derived from coinfection experiments in
tissue culture involving B/Yamagata/1/73 (B/Yam) and A/Aichi/2/68 viruses
in the presence of anti-A (H3N2) virus antibody. The growth of the mutant
influenza viruses in embryonated eggs of various ages were compared to
that of parental virus B/Yam, which possess a wild-type 281-amino-acid
NS1 protein. Eggs of 6, 10 and 14 days of age were inoculated with
approximately 103 pfuof B/Yam, B/201 or B/AWBY-234 viruses,
incubated at 35° C. for 2 days, and the viruses present in the
allantoic fluid were titrated by an HA assay.

[0100] Further, the attenuation characteristics of B/201 and B/AWBY-234
viruses were determined in mice. Groups of three BALB/c mice were
intranasally infected with 3×105 pfu of wild-type B/YAM or
B/201 and B/AWBY/234 mutant viruses, and the ability of these viruses to
replicate was determined by measuring viral titers in lungs at day 3
postinfection since wild-type B/Yam does not induce apparent signs of
disease in mice.

[0102] The results from the growth of the mutant and wild-type influenza B
viruses in embryonated chicken eggs, shown in Table 5, demonstrate that,
as in the case with influenza A viruses, a carboxy-terminal truncation of
the NS1 of influenza B virus is responsible for a lower replication yield
in older embryonated chicken eggs which mount an efficient IFN response.
This finding indicates that the NS1 of influenza B virus is also involved
in inhibiting the IFN responses of the host, and that deletions on the
NS1 gene of influenza B virus result in an attenuated phenotype.

[0103] The results from the replication experiments in mice are given in
Table 6. B/201 and B/AWBY-234 virus titers were approximately three logs
of magnitude lower that B/Yam titers, indicating that truncations of the
carboxy-terminal domain of the NS1 of influenza B virus are responsible
for an attenuated phenotype in mice.

8. PROTECTION AGAINST WILD-TYPE INFLUENZA VIRUS INFECTION IN MICE
IMMUNIZED WITH INFLUENZA A AND B VIRUSES CONTAINING DELETIONS IN THEIR
NS1 PROTEINS

[0104] In order to determine whether mice immunized with attenuated
influenza A and B viruses containing truncated NS1 proteins were
protected against challenge with their respective wild-type viruses the
following experiment was carried out. BALB/c mice were immunized
intranasally with A/NS1-99 virus and three weeks later they were infected
with 100 LD50 of wild-type influenza A/PR/8/34 virus. Immunized
animals were protected against death, while all control naive mice died
after the challenge (see Table 7). In a second experiment, BALB/c mice
were intranasally immunized with the influenza B viruses B/201 or
B/AWBY-234, expressing truncated NS1 proteins. Three weeks later the mice
were challenged with 3×105 pfu wild-type influenza B/Yam/1/73
virus. Since this strain of influenza B virus does not induce disease
symptoms in mice, the degree of protection was determined by measuring
virus titers in lungs at day 3 post-challenge. While naive control
animals had titers around 104 pfu/lung, viruses were not detected in
lungs of immunized animals (see Table 8). These findings suggest that
influenza A as well as influenza B viruses containing modified NS1 genes
are able to induce an immune response in mice which is fully protective
against subsequent wild-type virus challenge.

Induction of Type I Interferon in Embryonated Eggs Infected with delns1
Virus

[0105] The ability of delNS1 virus, an influenza A virus lacking the NS1
gene, to induce type I IFN secretion in embryonated chicken eggs was next
determined. For this purpose, groups of two 10-days-old embryonated
chicken eggs were infected with 5×103 pfu of delNS1 or
wild-type PR8 viruses. Eighteen hours postincubation at 37° C.,
the allantoic fluid was harvested and dialyzed against acid pH overnight,
to inactivate infectious viruses. After acid pH treatment, samples were
dialyzed against PBS, and they were tested for IFN activity by
determining the highest dilution with protective activity against VSV
infection (approximately 200 pfu) in CEF cells. The results shown in
Table 9 indicate that in the absence of NS1, influenza A viruses are
higher inducers of IFN.

[0106] Elimination of the IFN antagonist (NS1) gene from influenza A virus
may result in a virus with the ability to induce high levels of IFN. If
this is the case, delNS1 virus will "interfere" with the replication of
IFN-sensitive viruses. In order to test this possibility, Applicants
investigated the ability of delNS1 virus to inhibit the replication of
influenza A/WSN/33 (WSN) virus, a commonly used laboratory strain of
influenza virus, in eggs. As can be seen in FIG. 1, treatment with only 2
pfu of delNS1 virus was able to reduce the final titers of WSN virus in
the allantoic fluid by one log. In addition, treatment with
2×104 pfu of delNS1 virus resulted in practically complete
abrogation of WSN replication in eggs. DelNS1 virus was also able to
interfere with the replication in eggs of other influenza A virus strains
(H1N1 and H3N2), influenza B virus and a different virus such as Sendai
virus (FIG. 2).

[0107] Encouraged by these results, Applicants next determined the ability
of delNS1 virus to interfere with wild-type influenza virus replication
in mice. Although type I IFN treatment in tissue culture prevents
influenza A virus replication in vitro, treatment of mice with IFN is not
able to inhibit the replication of influenza viruses (Haller, 1981,
Current Top Microbiol Immunol 92:25-52). This is true for most inbred
strains of mice, except for A2G mice. A2G mice, as well as a significant
proportion of wild mice (approximately 75%), contain at least one intact
Mx1 allele, while most laboratory strains are Mx1-/- (Haller, 1986,
Current Top Microbiol Immunol 127:331-337). The Mx1 protein, which is a
homologue of the human MxA protein (Aebi, 1989, Mol. Cell. Biol.
11:5062), is a potent inhibitor of influenza virus replication (Haller,
1980, Nature 283:660). This protein is not constitutively expressed, but
its expression is transcriptionally induced by type I IFN. Thus, A2G mice
can be used to test the ability of IFN-inducers to stimulate an antiviral
response against influenza A viruses (Haller, 1981, Current Top Microbiol
Immunol 92:25-52).

[0108] Applicants intranasally infected eight 4-week-old A2G mice with
5×106 pfu of a highly pathogenic influenza A/PR/8/34 virus
isolate (Haller, 1981, Current Top Microbiol Immunol 92:25-52). Half of
the mice received an intranasal treatment with 5×106 pfu of
delNS1 at -24 h with respect to the PR8 infection. The other four mice
were treated with PBS. Body weight changes and survival was monitored.
These results demonstrate that delNS1 treatment was able to protect A2G
mice against influenza virus-induced death and body weight lost. The same
treatment was not effective in Mx1-/- mice indicating that the mechanism
of viral protection was Mx1, i.e. IFN, mediated.

11. EXAMPLE

Antitumor Properties of delns1 Virus in Mice

[0109] Given that type I IFN and/or inducers of type I IFN have been shown
to have antitumor activities (Belardelli and Gresser, 1996 Immunology
Today 17: 369-372; Qin et al., 1998, Proc. Natl. Acad. Sci. 95:
14411-14416), it is possible that treatment of tumors with delNS1 virus
might mediate tumor regression. Alternatively, delNS1 virus might have
oncolytic properties, i.e., it may be able to specifically grow in and
kill tumor cells, many of which are known to have deficiencies in the IFN
system. In order to test anti-tumor activity of delNS1 virus, the
following experiment was conducted using murine carcinoma cell line CT26
WT in a mouse tumor model for pulmonary metastasis (Restifo et al., 1998
Virology 249:89-97). 5×105 CT26 WT cells were injected
intravenously into twelve 6-weeks-old BALB/c mice. Half of the mice were
treated intranasally with 106 pfu of delNS1 virus every 24 hours at
days 1, 2 and 3 postinoculation. Twelve days after tumor injection, mice
were sacrificed and lung metastases were enumerated. As shown in Table
10, delNS1 treatment mediated a significant regression of murine
pulmonary metastases.

The NS1 Protein Inhibits the Translocation of IRF-3 During Influenza Virus
Infection

[0110] The results described herein suggest that the NS1 protein of
influenza virus is responsible for the inhibition of the type I IFN
response against the virus, and that mutations/deletions in this protein
result in attenuated viruses due to an enhanced IFN response during
infection. It is known that synthesis of type I IFN during viral
infection can be triggered by double-stranded RNA (dsRNA). IRF-3 is a
transcription factor which is usually found in an inactive form in the
cytoplasm of mammalian cells. Double-stranded RNA induces the
phosphorylation (activation) of the transcription factor IRF-3, resulting
in its translocation to the nucleus, where it induces transcription of
specific genes, including genes coding for type I IFN (Weaver et al.,
1998, Mol. Cell. Biol. 18:1359). In order to determine if NS1 of
influenza is acting on IRF-3, IRF-3 localization in CV1 cells infected
with wild-type PR8 or with delNS1 influenza A virus was monitored. FIG. 3
shows that IRF-3 translocation is minimal in PR8-infected cells (in fewer
than 10% of the cells). In contrast, approximately 90% of delNS1-infected
cells showed nuclear localization of IRF-3.

[0111] Strikingly, it was possible to partially inhibit the IRF-3
translocation in delNS1-infected cells by expressing NS1 from a plasmid
in trans. The results demonstrate that the NS1 of influenza A virus is
able to inhibit IRF-3 translocation in virus-infected cells. It is likely
that the NS1 of influenza virus prevents dsRNA-mediated activation of
IRF-3 by sequestering the dsRNA generated during viral infection, thus
resulting in an inhibition of IFN synthesis.

[0112] The present invention is not to be limited in scope by the specific
embodiments described which are intended as single illustrations of
individual aspects of the invention, and any constructs or viruses which
are functionally equivalent are within the scope of this invention.
Indeed, various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the art
from the foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of the appended
claims.

[0113] Various references are cited herein, the disclosures of which are
incorporated by reference in their entireties.